Broadly, the present invention involves a system and method for monitoring the stability of RNA and identifying agents capable of modulating RNA stability.
The relative stability of a mRNA is an important regulator of gene expression. The half-life of a mRNA plays a role in determining both the steady state level of expression as well as the rate of inducibility of a gene product. In general, many short-lived proteins are encoded by short-lived mRNAs. Several mRNAs that encode stable proteins, such as xcex1-globin, have also been shown to have extraordinarily long half-lives. Surveillance mechanisms are also used by the cell to identify and shorten the half-lives of mRNAs that contain nonsense codon mutations. Clearly, changes in the half-life of a mRNA can have dramatic consequences on cellular responses and function.
Little is known about mechanisms of mRNA turnover and stability in mammalian cells, but in vivo data are beginning to allow some generalizations about major pathways of mRNA turnover. The mRNA poly(A) tail can be progressively shortened throughout the lifetime of a mRNA in the cytoplasm. Controlling the rate of this deadenylation process appears to be a target for many factors that regulate mRNA stability. Once the poly(A) tail is shortened to approximately 50-100 bases, the body of the mRNA is degraded in a rapid fashion with no discernible intermediates. The process of translation also influences mRNA stability. Little is known, however, concerning the enzymes and regulatory components involved in mammalian mRNA turnover.
Several cis-acting elements have been shown to play a role in mRNA stability. Terminal (5xe2x80x2) cap and 3xe2x80x2-poly(A) structures and associated proteins are likely to protect the transcript from exonucleases. Several destabilizing as well as stabilizing elements located in the body of the mRNA have also been identified. The best characterized instability element is an A-U rich sequence (ARE) found in the 3xe2x80x2 untranslated region of many short-lived mRNAs. These AREs primarily consist of AUUUA (SEQ ID NO: 12) repeats or a related nonameric sequence. AREs have been shown to increase the rate of deadenylation and mRNA turnover in a translation-independent fashion. For example, proteins with AU-rich elements include many growth factor and cytokine mRNAs, such as c-fos, c-jun, c-myc TNFxcex1, GMCSF, IL1-15, and IFN-xcex2. Other stability elements include C-rich stabilizing elements, such as are found in the mRNAs of globin, collagen, lipoxygenase, and tyrosine hydroxylase. Still other mRNAs have as yet uncharacterized or poorly characterized sequence elements, for example, that have been identified by deletion analysis, e.g. VEGF mRNA.
Numerous proteins have been described that interact with some specificity with an ARE, bat their exact role in the process of mRNA turnover remains to be defined. For example, proteins which bind to the ARE described above include HuR and other ELAv family proteins, such as HuR (also called HuA), Hel-N1 (also called HuB), HuC and HuD; AUF 1 (four isoforms); tristetraprolin; AUH; TIA; TIAR; glyceraldebyde-3-phosphate; hnRNP C; hnRNP A1; AU-A; and AU-B. Many others have not been extensively characterized
Through the application of genetics, the mechanisms and factors involved in the turnover of mRNA in Saccharomyces cerevisiae are beginning to be identified. One major pathway of mRNA decay involves decapping followed by the action of a 5xe2x80x2-to-3xe2x80x2 exonuclease. Evidence has also been obtained for a role for 3xe2x80x2-to-5xe2x80x2 exonucleases in an alternative pathway. Functionally significant interactions between the cap structure and the 3xe2x80x2 poly(A) tail of yeast mRNAs have also been described. Several factors involved in the translation-dependent pathway of nonsense-codon-mediated decay have also been identified. Whether these observations are generally applicable to mammalian cells, however, remains to be established.
Mechanistic questions in mammalian cells are usually best approached using biochemical systems due to the inherent difficulties with mammalian cells as a genetic system. Thus, efforts have been made to develop in vitro systems to study mRNA stability and turnover. However, the presently available in vitro systems suffer from numerous limitations. For example, many suffer from poor data quality and a general lack of reproducibility that significantly limits their application. Another key problem is that most of these systems do not faithfully reproduce all aspects of mRNA stability. A significant difficulty in the development of these systems is to differentiate between random, non-specific RNA degradation and true, regulated mRNA turnover. The significance of all previous in vitro systems to the true in vivo process of mRNA stability, therefore, is unclear. To date, no in vitro mRNA stability system has been generally accepted in the field as valid and useful. Other problems that have been uncovered in presently available systems are that they usually involve a complicated extract protocol that is not generally reproducible by other laboratories in the field. Also, presently available systems can only be used to assess the stability of endogenous mRNAs, severely limiting their utility. Finally, the data quality obtained using such systems is highly variable, precluding their use in sensitive screening assays.
Accordingly, there exists a need for an in vitro RNA stability system is efficient and highly reproducible, and further, one which produces minimal to undetectable amounts of RNA degradation
A further need exists for an in vitro RNA stability system wherein deadenylation of an RNA transcript in the system should occur before general degradation of the mRNA body is observed. Also needed is an in vitro RNA stability system wherein degradation of the mRNA body occurs in an apparently highly processive fashion without detectable intermediates, and further, the regulation of the rate of overall deadenylation and degradation should be observed in a sequence-specific manner. Such a system should be applicable to exogenous RNAs and allow ease of experimental manipulation.
The citation of any reference herein should not be construed as an admission that such reference is available as xe2x80x9cPrior Artxe2x80x9d to the instant application.
In accordance with the present invention, an in vitro system for modulating the stability and turnover of an RNA molecule is provided which models RNA processing in vivo. Thus, the present invention permits high throughput screening of compounds/macromolecules that modulate the stability of eukaryotic RNAs in order to identify and design drugs to affect the expression of selected transcripts, as well as to aid in the characterization of endogenous proteins and other macromolecules involved in mRNA stability. The in vitro system of the present invention is useful as a diagnostic aid for determining the molecular defect in selective disease alleles; development of in vitro mRNA stability systems for other eukaryotic organisms including parasites and fungi which should lead to novel drug discovery; and improving gene delivery systems by using the system to identify factors and RNA sequences that affect RNA stability.
Broadly, the present invention extends to an in vitro system capable of recapitulating regulated RNA turnover of an exogenously added preselected target RNA sequence, the system comprising a cell extract and a target RNA sequence. In a non-limiting example of the system described herein, the regulated RNA turnover is AU-rich element regulated RNA turnover or C-rich element regulated RNA turnover.
The cell extract of the system of the present invention is isolated from lysed eukaryotic cells or tissues; the cell extract may be obtained for example from a cell line, such as HeLa cells or a T cell line, but the invention is not so limited. The cell extract may be prepared from cells comprising foreign nucleic acid, such as those that are infected, stably transfected, or transiently transfected. The cell extract may be partially purified.
In one embodiment of the invention, the cell extract may be depleted of activity of proteins that bind polyadenylate. The depletion of activity of proteins that bind polyadenylate from the cell extract may be achieved by any of a number of methods, for example, the addition to the system of polyadenylate competitor RNA; the sequestration of proteins that bind polyadenylate; the addition of a proteinase that inactivates a protein that bind to polyadenylate; or addition of an agent that prevents the interaction between polyadenylate and an endogenous macromolecule that binds to polyadenylate, to name a few. As further examples of the methods for sequestration of proteins that bind polyadenylate, it may be achieved by such non-imuniting procedures as the treatment of the extract with an material that depletes macromolecules that bind polyadenylate, such as antibodies to proteins that bind polyadenylate, polyadenylate, and the combination. The material may be attached to a matrix. Other methods to achieve the depletion of the activity of proteins that bind polyadenylate may be used.
The target RNA sequence used in the system may be, by way of non-limiting examples, synthetic RNA, naturally occurring RNA, messenger RNA, chemically modified RNA, or RNA-DNA derivatives. The target RNA sequence may have a 5xe2x80x2 cap and a 3xe2x80x2 polyadenylate sequence. The target RNA sequence may be unlabeled target RNA sequence, labeled target RNA sequence, or a the combination of both. The labeled RNA sequence may be labeled with a moiety such as, but not limited to a fluorescent moiety, a visible moiety, a radioactive moiety, a ligand, and a combination of fluorescent and quenching moieties. Other moieties and means for labeling RNA are embraced herein.
The system of the present invention may additionally include exogenously added nucleotide triphosphate; ATP is preferred. It may also include a reaction enhancer to enhance the interaction between the various components present in the system, for example, polymers such as but not limited to polyvinyl alcohol, polyvinylpyrrolidone and dextran; polyvinyl alcohol is preferred.
The present invention is also directed to a method for identifying agents capable of modulating the stability of a target RNA sequence. The method is carried out by preparing the system described above which includes the cell extract depleted of activity of proteins that bind polyadenylate and the target RNA sequence; introducing into the aforesaid system an agent to be tested; determining the extent of turnover of the target RNA sequence by, for example, determining the extent of degradation of the labeled target RNA; and then identifying an agent which is able to modulate the extent of RNA turnover as capable of modulating the stability of the target RNA sequence.
The method described above may additionally include nucleotide triphosphate, ATP being preferred. The agent to be tested may be, but is not limited to, an RNA stability modifying molecule. The non-limiting selection of the types of target RNA sequence and the non-limiting types of labels useful for the RNA as described hereinabove.
The method of the present invention is useful for identifying agents which can either increase or decrease the stability of said target RNA sequence. Such agents may be capable of modulating the activity of an RNA binding molecule such as, but not limited to, C-rich element binding proteins and AU rich element binding proteins, examples of the latter including HuR and other ELAv family proteins, such as HuR, Hel-N1, HuC and HuD; AUl 1; tristetraprolin; AUH; TIA; TIAR; glyceraldehyde-3-phosphate; hnRNP C; hnRNP A1; AU-A; and AU-B. This list is provided as illustrative of the types of molecules that may be evaluated in the present invention, but is by no means limiting.
In a further embodiment of the present invention, a method is provided for identifying an agent that is capable of modulating the stability of a target RNA sequence in the presence of an exogenously added RNA stability modifier or RNA binding macromolecule. Non-limiting examples of such molecules are described above. The method is carried out by preparing the system described above which includes the cell extract can be depleted of activity of proteins that bind polyadenylate and the target RNA sequence; introducing into the aforesaid system the exogenously added RNA stability modifier or binding macromolecule and the agent to be tested; determining the extent of turnover of the target RNA sequence by, for example, determining the extent of degradation of the labeled target RNA; and then identifying an agent able to modulating the extent of the RNA turnover as capable of modulating the stability of the target RNA sequence in the presence of the exogenously added RNA stability modifier.
The non-limiting selection of the components of this method are as described above. The aforementioned method is useful, for example, when the RNA stability modifier decreases the stability of said target RNA sequence, and the agent to be identified increases the stability of the target RNA sequence that is decreased by the RNA stability modifier in addition, the method is useful when the RNA stability modifier increases the stability of the target RNA sequence, and the agent to be identified decreases the stability of the target RNA sequence that is increased by the RNA stability modifier. Non-limiting examples of RNA stability modifiers include C-rich element binding proteins, and AU rich element binding proteins, examples of AU rich element binding proteins, including HuR and other ELAv family proteins, such as HuR, Hel-N1, HuC and HuD; AUF1; tristetraprolin; AUH; TIA; TIAR; glyceraldehyde-3-phosphate; hnRNP C; hnRNP A1; AU-A; and AU-B. This list is provided as illustrative of the types of molecules that may be evaluated in the present invention, but is by no means limiting.
The present invention is further directed to a method for identifying an agent capable of modulating the deadenylation of a target RNA sequence comprising preparing the system described above in the absence of nucleotide triphosphate, such as ATP; introducing an agent into the system; and monitoring the deadenylation of the target RNA sequence. Furthermore, the invention is also directed towards a method for identifying an agent capable of modulating the deadenylation and degradation of a target RNA sequence comprising preparing the system described herein in the presence of ATP; introducing the agent into the system; and monitoring the deadenylation and degradation of the target RNA sequence. These embodiments may also be carried out in the presence of an RNA stability modifier or RNA binding macromolecule to determine the ability of the agent to modulate the effect of the modulator or binding molecule on RNA stability.
It is a further aspect of the present invention to provide a method for identifying an agent capable of modulating cell growth or cell differentiation in a mammal comprising determining the ability of said agent to modulate the stability of a target RNA sequence involved in the modulation of cell growth or differentiation in accordance with the methods described above. The agents capable of modulating cell growth or cell differentiation may intervene in such physiological processes as cellular transformation and immune dysregulation, but the invention is not so limiting.
It is yet a further aspect of the present invention to provide a method for identifying, characterizing and isolating an endogenous molecule suspected of participating in the deadenylation or degradation of RNA or regulation thereof comprising preparing the system described hereinabove; introducing a protein suspected of participating in the regulation of RNA turnover into said system; and monitoring the stability of the target RNA sequence. The endogenous molecule suspected of participating in the deadenylation and/or degradation of RNA or regulation may be protein or RNA.
In another embodiment of the invention, a method is provided for identifying an agent capable of modulating the degradation a target RNA sequence in the absence of deadenylation comprising providing a cell extract in the presence of a nucleotide triphosphate; introducing said agent into said cell extract; and monitoring the degradation of said target RNA sequence in said extract.
A further aspect of the present invention is directed to a kit for monitoring the stability of a preselected target RNA sequence under conditions capable of recapitulating regulated RNA turnover. The kit comprises a cell extract that optionally may be depleted of activity of proteins that bind polyadenylate; other reagents; and directions for use. The kit may further comprise nucleotide triphospliates, a reaction enhancer, or both.
Accordingly, it is an object of the invention to provide a system for modulating the stability and turnover of an RNA molecule in vitro, which permits a skilled artisan to study the turnover generally, or deadenylation and degradation specifically, of an RNA transcript, and screen drugs which can modulate the stability and turnover of an RNA transcript. The turnover may be in the absence or presence of exogenously added RNA stability modulators, or permit the study of the role of endogenous molecules in RNA turnover.
It is another embodiment of the invention to provide a kit that a skilled artisan can readily use to modulate the stability and turnover of an RNA molecule in vitro, and investigate the aforementioned agents.